title: Thiazole orange-induced c-di-GMP quadruplex formation facilitates a simple fluorescent detection of this ubiquitous biofilm regulating molecule.
abstract: Recently, there has been an explosion of research activities in the cyclic dinucleotides field. Cyclic dinucleotides, such as c-di-GMP and c-di-AMP, have been shown to regulate bacterial virulence and biofilm formation. c-di-GMP can exist in different aggregate forms, and it has been demonstrated that the polymorphism of c-di-GMP is influenced by the nature of cation that is present in solution. In previous work, polymorphism of c-di-GMP could only be demonstrated at hundreds of micromolar concentrations of the dinucleotide, and it has been a matter of debate if polymorphism of c-di-GMP exists under in vivo conditions. In this Article, we demonstrate that c-di-GMP can form G-quadruplexes at low micromolar concentrations when aromatic molecules such as thiazole orange template the quadruplex formation. We then use this property of aromatic molecule-induced G-quadruplex formation of c-di-GMP to design a thiazole orange-based fluorescent detection of this important signaling molecule. We determine, using this thiazole orange assay on a crude bacterial cell lysate, that WspR D70E (a constitutively activated diguanylate cyclase) is functional in vivo when overexpressed in E. Coli . The intracellular concentration of c-di-GMP in an E. Coli cell that is overexpressed with WspR D70E is very high and can reach 2.92 mM.
authors.affiliation: Department of Chemistry and Biochemistry, University of Maryland, College Park, Maryland 20742, USA.
authors.forename: Shizuka
authors.initials: S
authors.lastname: Nakayama
authors.forename: Ilana
authors.initials: I
authors.lastname: Kelsey
authors.forename: Jingxin
authors.initials: J
authors.lastname: Wang
authors.forename: Kevin
authors.initials: K
authors.lastname: Roelofs
authors.forename: Bogdan
authors.initials: B
authors.lastname: Stefane
authors.forename: Yiling
authors.initials: Y
authors.lastname: Luo
authors.forename: Vincent T
authors.initials: VT
authors.lastname: Lee
authors.forename: Herman O
authors.initials: HO
authors.lastname: Sintim
mesh_terms: D052160:Benzothiazoles / Q000737:chemistry*; D018441:Biofilms; D006152:Cyclic GMP / Q000031:analogs & derivatives* / Q000032:analysis / Q000138:chemical synthesis / Q000737:chemistry; D004926:Escherichia coli / Q000737:chemistry / Q000166:cytology / Q000378:metabolism; D005453:Fluorescence; D054856:G-Quadruplexes; D008968:Molecular Conformation; D011804:Quinolines / Q000737:chemistry*
chemical_list: D052160:Benzothiazoles; D011804:Quinolines; C051000:thiazole orange; C062025:bis(3',5')-cyclic diguanylic acid; D006152:Cyclic GMP